1. Field of the Invention
The present invention relates generally to atomic force microscopy (AFM) and to apparatus and methods for measuring intermolecular interactions such as receptor/ligand, antibody/antigen and DNA interactions efficiently by atomic force microscopy.
2. Description of the Related Art
In conventional atomic force microscopy, a sample is scanned with a fine-tipped probe mounted on a cantilever, and deflections of the probe tip as it passes across the sample are measured to determine the topography of the sample. Deflections in the probe tip as it passes along the sample surface may be monitored by various methods, including optical reflection, interferometry, and piezoelectric strain gauge methods. See, for example, U.S. Pat. No. Re. 33,387 to Binnig, U.S. Pat. No. 5,144,833 to Amer et al, U.S. Pat. No. 5,463,897 to Prater et al, U.S. Pat. No. Re. 34,489 to Hansma et al and U.S. Pat. No. 5,260,824 to Okada et al, all of the above incorporated herein by reference.
In recent years, atomic force microscopy has been used to measure interfacial properties and intermolecular interactions such as elasticity, friction, adhesion, receptor/ligand interactions, and antibody/antigen interactions between individual molecules. To measure binding interactions between complimentary ligands and receptors, the probe tip of a cantilever and a sample surface may be chemically modified by various means to attach the complementary ligands and receptors to each. (For convenience, the compound or compounds immobilized on the cantilever or similar structures will be referred to herein as "reference" compounds and the compound or compounds immobilized on the sample support member or sample surface will be referred to herein as "sample" compounds.) The probe tip and sample surface may then be brought into proximity or into contact so that a ligand and a receptor interact or bond. When the probe tip and sample surface are then separated, the ligand-receptor bond breaks and the strength of the interaction force between the ligand and the receptor may be measured. The use of atomic force microscopy to study intermolecular forces is described, for example, in the following patents and publications, incorporated herein by reference: U.S. Pat. No. 5,363,697 to Nakagawa; U.S. Pat. No. 5,372,930 to Colton et al; Florin E.-L. et al, "Adhesion Forces Between Individual Ligand-Receptor Pairs" Science 264 (1994). pp 415-417; Lee, G.U et al, "Sensing Discrete Streptavidin-Biotin Interactions with Atomic Force Microscopy" Langmuir, vol. 10(2), (1994) pp 354-357; Dammer U. et al "Specific Antigen/Antibody Interactions Measured by Force Microscopy" Biophysical Journal Vol. 70 (May 1996) pp 2437-2441; Chilikoti A. et al, "The Relationship Between Ligand-Binding Thermodynamics and Protein-Ligand Interaction Forces Measured by Atomic Force Microscopy" Biophysical Journal Vol. 69 (November 1995) pp 2125-2130; Allen S. et al, "Detection of Antigen-Antibody Binding Events with the Atomic Force Microscope" Biochemistry, Vol. 36, No. 24 (1997) pp 7457-7463; and Moy V.T. et al, "Adhesive Forces Between Ligand and Receptor Measured by AFM" Colloids and Surfaces A: Physicochemical and Engineering Aspects 93 (1994) pp 343-348. If interactions between molecules are studied in liquids, the experimental conditions, such as pH, buffer/ionic concentration, buffer/ionic species, etc. may be varied to determine the effect that these have on the forces of interaction.
Atomic force microscopy has great potential for use in screening arrays of compounds, such as libraries of compounds produced by combinatorial methods, to identify useful ligand/receptor interactions and to discover useful drugs. Modem methods of combinatorial chemistry, parallel synthesis, and microlithography make it possible to produce large, compact libraries of chemical analogues in spatially addressable arrays. Atomic force microscopy with chemically modified probe tips provides a way of screening these arrays. However, the equipment and techniques currently used for atomic force microscopy are not well suited for repetitive measurements or efficient high-volume screening. In particular, chemically modified cantilever probe tips are fragile and easily damaged or inactivated. In a typical chemically modified cantilever probe tip, only the molecules that are bound to the very apex of the tip are available for force interactions with a substrate. The crucial area of the probe tip is typically very small and the number of molecules bound thereon is very few; if anything happens to damage those few molecules or to block access to, or to otherwise inactivate that small area of the probe tip, then the probe tip is rendered useless and must be replaced. Replacing the probe tip usually requires replacing the entire cantilever, a procedure that is expensive and time-consuming. Moreover, the typical cantilever probe tip has room for only one reference compound to be immobilized on the tip, so the cantilever must be replaced or modified whenever it is desired to use a different reference compound.